Dendritic cells (DCs) and macrophages are capable of acquiring and then displaying peptides from external antigens through a process called cross presentation. This process is the key mechanism that allows the immune system to recognize and then mobilize a CD8 T cell response to cancers, many viral infections and intracellular pathogens. Consequently, this pathway is important for immune surveillance and is an attractive target to enable vaccines to elicit CD8 T cell immunity. Given this, it is important to elucidate te mechanisms that allow DCs to carry out this critical function and this is the overall goal of this grant. To this end, we propose to characterize and elucidate the function of two key and previously unsuspected novel cross presentation genes that we discovered in an unbiased, genome-wide forward genetic screen. Our first Aim will elucidate the role of Rab39A in cross presentation. Very interestingly, while Rab39A is required for cross presentation but not conventional MHC I or MHC II presentation. Rab proteins are GTPases that control the formation, content, trafficking and ultimate fusion of vesicles in cells and thereby determine the composition of endocytic compartments. Our favored hypothesis is that Rab39A is functioning to help form a specialized XPT compartment in DCs by trafficking essential components of the XPT pathway. Furthermore, given their role in determining the composition of vesicles, Rabs and their recruited effectors serve as markers to identify and isolate unique endocytic compartments (e.g. early endosomes, late endosomes, lysosomes, melanosomes, etc.). In this context, and very interestingly, Rab39A is only present in a subset of phagosomes in DCs. Given this, we also hypothesize that Rab39A will mark the XPT compartment. The importance of these hypotheses is that Rab39A could solve the mystery as to how MHC I and/or other components from the ER get to phagosomes; clarify where XPT is carried out and its relationship to other pathways such as MHC II; and/or allow the characterization of the Rab39A+ compartment to identify novel components in the pathway. This Aim will test these hypotheses and more generally explore and elucidate the role of Rab39A in XPT. Our second Aim will elucidate the role of Loc547349 (Loc) in cross presentation. Our preliminary data show that this molecule is required for cross presentation but not classical MHC I or MHC II antigen presentation. Very interestingly, Loc is an MHC-encoded MHC I-like molecule of unknown function. This discovery is very intriguing because many of the key genes for MHC I and MHC II antigen presentation are encoded in the MHC locus. Moreover, there is a similar molecule, HLA-DM, that operates as a peptide editor in the MHC II pathway and for which up until now has not had a clear counterpart in the MHC I pathway, yet there is a theoretical need for such an activity for cross presentation. Our favored hypotheses are that Loc functions in cross presentation to help traffic MHC I molecules to endocytic compartments, stabilize them, and/or load them with peptides. The goal of this aim is to test this hypothesis as well as other potential functions and elucidate the role of Loc in cross presentation.